Structure-function analysis of DNA polymerase: Purification, characterization and in vitro mutagenesis of PRD1 DNA polymerase.
Committee ChairIto, Junetsu
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PublisherThe University of Arizona.
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AbstractA small lipid-containing bacteriophage PRD1 encodes a DNA polymerase that utilizes a protein primer for the initiation of DNA replication. The purification of PRD1 DNA polymerase has been hampered by the insolubility of the overexpressed enzyme in E. coli cells. A simple and rapid procedure for purification of the overexpressed PRD1 DNA polymerase has been developed. This method is based on guanidine hydrochloride denaturation and renaturation of the insoluble PRD1 DNA polymerase overexpressed in E. coli containing recombinant plasmid pEJG. The purified DNA polymerase was extensively characterized and found to be indistinguishable from the normal soluble PRD1 DNA polymerase as judged by enzymatic properties. These properties include: protein-primed initiation of PRD1 DNA replication, strand-displacement DNA synthesis, DNA polymerase processivity, 3' to 5' exonuclease activity and filling-in repair type DNA synthesis. Furthermore, the kinetic parameters determined for dNTPs and primer-terminus were of the same order of magnitude. The availability of a simple purification procedure for PRD1 DNA polymerase should permit detailed structure-function analysis of this enzyme. All known family B DNA polymerases contain a conserved region of amino acids, KX₆₋₇YG, which appears to be correspond to the "finger" alpha helix O of the Klenow fragment of E. coli DNA polymerase I, a family A DNA polymerase. A site-directed mutagenesis study has been applied to access the functional role of the invariant amino acid lysine-340 of PRD1 DNA polymerase. Mutant DNA polymerases were overexpressed and purified to near homogeneity. The results showed that the modification of the lysine-340 of PRD1 DNA polymerase abolishes the polymerase activity without affecting the 3' to 5' exonuclease activity. Site-directed mutagenesis studies revealed that residues important for the 3' to 5' exonuclease activity, particularly metal binding ligands for the Klenow fragment, are all conserved in PRD1 DNA polymerase as well. Although PRD1 DNA polymerase has a smaller 3' to 5' exonuclease domain, active sites appear to be very similar to those of the Klenow fragment. Moreover, the metal binding ligands were also found to be important for the strand-displacement activity, a unique feature of PRD1 DNA polymerase.
Degree ProgramMicrobiology and Immunology